1. Field of Invention
The present invention relates to a method of producing protein or peptide by the culture of recombinant cells.
2. Related Art
Numerous useful substances are produced by culturing microorganisms or animal cells.
Although the production of useful substances using microorganisms such as E. coli is advantageous in terms of the amount produced per medium or productivity per cell, such a method cannot be applied in cases wherein the target protein has a high molecular weight and has a too complex steric structure to be refolded, or in cases of substances whose physiological activity is exhibited only after modification of such as sugar chains.
Animal cells are typically used to produce such proteins. Examples of known animal cells include CHO-K1 (Chinese hamster ovary cell: ATCC CCL-61), CHO-K1-derived dihydro folate reductase (DHFR) gene-deficient line, hybridomas constructed by fusing a parent cell (myeloma, etc.) with a useful substance-producing normal cell (lymphocyte, etc.), C127I (mouse mammary tumor cells: ATCC CRL-1616), BHK-21(C-13) (baby hamster kidney cell: ATCC CCL-10) and Vero (African green monkey kidney cell: ATCC CCL-81).
However, shortcomings of animals cells when compared with microorganisms include a slow growth rate, expensive media and low production per medium and productivity per cell. The use of large-scale culture vessels, basic culture conditions (incubation temperature, dissolved oxygen concentration, pH, etc.), media (serum-free media, protein-free media, etc.), medium additives (butyric acid, dimethylsulfoxide (DMSO), hydrocortizone, etc.) and increased density of cultured cells are being examined as ways of dealing with these shortcomings.
In the culturing of animals cells, the incubation temperature is almost always 37.degree. C. However, since incubation temperature is considered to have an effect on cell growth as well as the metabolism of various substances, optimum temperature is an indispensable element of substance production by cell culturing.
The effect of incubation temperature was first studied in the 1970's in the production of interferon. In addition, there are also several reports of studies in monoclonal antibody-producing hybridomas starting in the 1980's.
With respect to the production of interferon, by treating normal fibroblasts, Burkitt's lymphoma cells (Namalwa cells), rabbit kidney cells (RK13), other lymphoblast-like cells and so forth with various chemicals such as poly I (polyinosinic acid), poly C (polycytidylic acid), cycloheximide, actinomycin D and butyric acid, or viruses such as Sendai virus, it is possible to create a system that induces production of interferon. It is reported that in this system, the amount of interferon can be increased by lowering the incubation temperature (Proc. Nat. Acad. Sci. USA, Vol. 70, No. 12, Part II, pp.3909-3913, 1973; Japan J. Microbiol., Vol. 18(3), 217-222, 1974; Antimicrob. Agents Chemother., Vol. 18, No. 1, p.130-136, 1980; Proceedings of the Society for Experimental Biology and Medicine, 170, 155-159, 1982; J. Interferon Res. 11, Suppl. 1, S211, 1991; and, Japanese Unexamined Patent Publication No. 7-31495).
However, since the methods indicated there require complex procedures for inducing production, large-volume production on an industrial scale is difficult. Moreover, this is not a culturing method that can be universally applied to animal cells.
With respect to monoclonal antibody-producing hybridomas, it has been reported that in the case of lowering the incubation temperature, although a high viability is maintained for a long time and glucose consumption is reduced, monoclonal antibody productivity decreases (Biotechnology and Bioengineering, Vol. 37, pp.292-295, 1991). It has also been reported that although the number of cells in the G1 stage of the cell cycle increases, antibody productivity per cell does not change, with maximum cell growth and maximum antibody production occurring during culture at 37.degree. C. (Biotechnology and Bioengineering, Vol. 40, pp.427-431, 1992), thus indicating different interpretations depending on the cell line used.
Antibody production in hybridomas is thought to be affected by the properties of the parent cell line such as myeloma as well as the antibody-producing lymphocytes that are fused with it. Thus results are thought to differ depending on the cell line used.
On the other hand, in CHO cells that are commonly used as host cells for gene recombination, although there are reports that the optimum temperature for cell growth is 37.degree. C. (Journal of Biotechnology, 15, 101-111, 1990), the optimum temperature for substance production is completely unknown.
In addition, with respect to the effect of incubation temperature on gene recombinant cells, although there is a report that a temperature-sensitive mutant strain derived from CHO cells (optimum temperature for cell growth: 34.degree. C., for substance production: 39.degree. C.) was constructed and applied for substance production (Biotechnology and Bioengineering, Vol. 42, pp.1029-1036, 1993), since a temperature-sensitive strain was used, no assumptions can be made regarding the effect of incubation temperature on normal gene recombinant cells.
Thus, there is currently very little information available relating to the effect of incubation temperature on gene recombinant cells. In addition, the previously illustrated reports involved studies of the effects of incubation temperature from the aspect of cell growth or substance productivity. However, when considering substance production by cell culturing, since it is also necessary to consider the following purification process as well as the culturing itself, studies should focus on more comprehensive aspects including consumption of media components, contaminating proteins and so forth in addition to cell growth and substance productivity. Thus, with respect to animal cells in general, findings regarding the effects of incubation temperature can be said to be insufficient at present.